PROJECT SUMMARY/ABSTRACT Triple Negative Breast Cancer (TNBC) is a highly aggressive subtype of breast cancer which exhibits both enhanced angiogenesis and vasculogenic mimicry or VM, i.e., the direct formation and lining of vascular channels by tumor cells. While the expression of epidermal growth factor receptor (EGFR, HER1) is reported in greater than 60% of TNBC and is similarly expressed in 65% to 72% of basal-like tumors, no proven targeted therapy is currently available for the treatment of TNBC. Cetuximab (Erbitux), a chimeric monoclonal antibody targeting EGFR, elicits little response as single-agent therapy in the setting of advanced TNBC. In the present study, the combined inhibition of angiogenesis and VM is proposed as a potentially superior therapeutic strategy for inhibiting TNBC growth and metastasis. Our approach utilizes a fusion protein that was generated by linking an anti-EGFR MAb as a targeted delivery entity of two molecules of a mutant form of human endostatin, huEndoP125A (where alanine substitution of Proline 125 significantly increases the anti-angiogenic potency of endostatin). The Anti-EGFR antibody-coupled-huEndo-P125A fusion (anti-EGFR-huEndo-P125A) containing bivalent or ?dimeric? huEndo-P125A, thus generated, exhibits potent inhibition of angiogenesis by endothelial cells. More interestingly, it also demonstrates profound inhibition of VM not seen with parental antibody nor with wild-type endostatin, or huEndo-P125A alone, or when combined with cetuximab (without fusion) suggesting that the bivalent or dimeric endo-P125A in fusion is highly essential for VM inhibition. The fusion is differentiated from anti-VEGF antibody, bevacizumab which did not inhibit TNBC VM, suggesting the fusion also has mechanisms of action distinct from targeting of the VEGF pathway. We also observed a consistent decrease in total ?-catenin levels following fusion treatment of MDA-MB-231 TNBC cells which is consistent with inhibition of Wnt signaling pathway, while endostatin had minimal effects at similar molar concentrations. Inhibition of VM by the anti-EGFR-huEndo-P125A fusion proteins was also associated with significantly reduced tumor cell motility. We have now generated a stable CHO cell pool with fusion production yield of >200mg/liter and have optimized the purification methods that show >95% purity of monomeric fusion protein that is stable at both 40 and -200 for several months. In an initial experiment, the fusion has shown am markedly enhanced inhibitory effect on inhibition of TNBC lung metastasis compared to cetuximab. These promising preliminary results provide the basis for accelerated testing of the potential therapeutic utility of dual inhibition of both angiogenesis and VM on TNBC growth and metastasis in vivo to generate pre-clinical data that will lead to therapeutic testing in human TNBC patients. Due to the striking in vitro and encouraging preliminary in vivo results, and with the availability of cell line for production of large quantity of the fusion protein, we will focus on TNBC as an unmet need and extend our work to optimize the effects of the fusion by itself or in combination with approved anti-angiogenics such as bevacizumab and/or chemotherapy agent(s) on TNBC growth and metastasis. The fusion will also be tested for its ability to mediate effective ADCC. We anticipate that the multifunctional effects of anti-angiogenesis, VM inhibition and ADCC will render ??EGFR-IgG1-huEndo-P125A a breakthrough therapeutic for TNBC and other EGFR+ malignancies.